Because cancer is a heterogenous disease at the clinical, histological, and molecular levels and because tumor phenotype often changes as tumors develop, the efficacy of established treatments vary quite significantly among individual patients. This is particularly true for therapies that target specific tumor biomarkers. A useful strategy to guide the development of individualized, more efficient treatment strategies is molecular profiling of tumors. However, current tumor profiling methods are suboptimal: they require multiple biopsies, their results are often compromised by sampling errors and inconsistencies in pathologists' interpretations; also, current methods are not applicable to disseminated micrometastases. A more accurate method of profiling individual tumors is thus clearly needed, as it would help to improve the efficacy of targeted treatments in potentially responsive patients and avoid such treatments in patients unlikely to respond. Toward this end, the objective of this proposed research is to develop and test a new generation of targeted radiotracers for use in positron emission tomography (PET) imaging of tumors' molecular signatures. The proposed PET strategy will facilitate detection of target tumors and enable accurate, noninvasive quantitative measurement of intratumoral levels of an established tumor biomarker that predicts disease progression and responsiveness to targeted treatments. We propose using a novel molecular prototype for these radiotracers in combination with the short-lived radioisotopes to produce data quickly, thereby reducing the burden on patients and accelerating the diagnosis-treatment cycle. We hypothesize that Designed Ankyrin Repeat Proteins (DARPins), a novel class of rationally designed proteins, represent a desirable advanced molecular platform for tracer design. DARPins' unique biological properties make them an excellent alternative to antibodies (Ab) and Ab derivatives that are traditionally used for this purpose despite their known limitations. We will test our hypothesis and achieve our objective by pursuing two specific aims: Specific aim 1. Develop and characterize in vitro DARPin-based radiotracers for PET imaging of human epidermal growth factor receptor-2 (Her2). Specific aim 2. Demonstrate the specificity of DARPin-enabled PET imaging for Her2 and determine its detection limits in an orthotopic model of cancer. This work will facilitate the development of personalized treatment plans best suited for individual cancer patients and thus yield significant improvements in treatment efficacy and cost efficiency.